August 7, 2020

BACKGROUND

Biologics are enormously useful technologies

(Sanchez-Garcia et al. 2016)

Why fungi?

(Love, Dalvie, and Love 2017)

Challenges limit bioproduction

(Wang, Huang, and Nielsen 2017)

Secretion challenges: Translation

(Nelson and Cox 2017)

Secretion challenges: ER Trafficking

(Reece et al. 2015)

Secretion challenges: Folding

(Klaips, Jayaraj, and Hartl 2018)

Secretion challenges: Degradation

(Hampton 2000)

QUANTIFICATION FOR RATIONAL DESIGN

Approaches that improve bioproduction

A. Engineer secretory system

B. Designing signal peptides

C. Depleting competing resources

(Huang et al. 2018; Mori et al. 2015; Kallehauge et al. 2017)

Using data science for rational bioproduction

(Dykstra, Rousu, and Arvas 2017)

Quantifying demands

Ribo-Seq for quantification

(“BRAR LAB — the Brar and Ünal Labs,” n.d.; McGlincy and Ingolia 2017)

Separating translation events in the cytosol and ER

Data processing and alignment

(Martin 2011; “HISAT2,” n.d.; Li et al. 2009)

Resolution and counting codon reads

(Lareau et al. 2014)

EXPERIMENTAL CHALLENGES

Problem: Multimapping

(Taggart and Li, n.d.)

Correcting biases and quantifying demand

Problem: Annotations

Example: Misannotated translational start site for TIF1

Description
ATP-dependent RNA helicase and translation initiation factor

Predicted Location
Cytoplasm

Membrane Enriched?
FALSE

Incorrect annotation

Problem: Annotations

Example: Misannotated translational start site for TIF1

Description
ATP-dependent RNA helicase and translation initiation factor

Predicted Location
Cytoplasm

Membrane Enriched?
FALSE

Correct annotation

Modifying annotations

Comparing annotations

Annotation * Total sequences Homologous sequences Sequence differences
Current study 5,329
GS115 (PRJNA304976) 5,064 5,035 514
GS115 (PRJEA37871) 5,040 5,100 697
CBS7435 (PRJEA62483) 5,291 5,198 604
* Parenthesis contains NCBI accession numbers

QUANTIFYING DEMANDS

Biosynthetic demands by ontology

Nascent chains (%) Genes (n)
Ontological Functions
Translation, Ribosomal Structure and Biogenesis 44.0% 366
Function Unknown 11.0% 1602
Post-Translational Modification, Protein Turnover and Chaperones 9.0% 409
Energy Production and Conversion 8.0% 207
Intracellular Trafficking, Secretion and Vesicular Transport 4.0% 382
Carbohydrate Transport and Metabolism 3.0% 218
Cell Wall/Membrane/Envelope Biogenesis 3.0% 85
Amino Acid Transport and Metabolism 3.0% 191
Transcription 2.0% 355
RNA Processing and Modification 2.0% 242
Predicted Features
Signal Sequences 8% 266
GPI Anchors 79%* 117
Transmembrane Domains 7% 960
* Percentage of signal sequences that contain GPI anchors

Translation in the cytosol and membranes

Comparing demands in K. phaffii

ER demands by ontology

Genes (n) Nascent chains (%) Ribosomes (%)
Cotranslationally Targeted
Function Unknown 261 7.90% 10.70%
Cell Wall/Membrane/Envelope Biogenesis 41 7.40% 11.90%
Post-Translational Modification, Protein Turnover and Chaperones 89 6.80% 11.60%
Carbohydrate Transport and Metabolism 114 6.60% 9.50%
Intracellular Trafficking, Secretion and Vesicular Transport 95 6.30% 7.40%
Inorganic Ion Transport and Metabolism 82 4.90% 9.70%
Lipid Transport and Metabolism 72 3.80% 5.40%
Posttranslationally Targeted
Function Unknown 30 33.40% 9.60%
Cell Wall/Membrane/Envelope Biogenesis 10 14.20% 8.90%
Post-Translational Modification, Protein Turnover and Chaperones 5 0.30% 0.20%

Comparison of nascent chains produced

Comparison of ribosomes used

Possible targets for K. phaffii

Highest Expressed Cotranslationally Targeted Proteins
Gene cTPM Length Description
PST1 2374 418 Cell wall protein
PMA1 1968 896 Plasma membrane H+-ATPase
KAR2 1325 678 Protein import-associated ATPase
GAS1 772 538 1,3-beta-glucanosyltransferase (isoform 1)
ERG5 314 528 C-22 sterol desaturase
SIT1 293 633 Ferrioxamine B transporter (isoform 1)
AOA68981.1 250 587 Uncharacterized protein
OST1 226 463 ER oligosaccharyltransferase complex subunit alpha
WBP1 203 431 ER oligosaccharyltransferase complex subunit beta
SEC61 195 478 SEC61 translocation complex subunit
HNM1 177 541 Choline/ethanolamine transporter
MUP1 174 562 Methionine permease (isoform 1)
PMT4 173 741 Protein O-mannosyltransferase
PMT2 170 750 Protein O-mannosyltransferase
VPS70 168 838 Uncharacterized vacuolar protein sorting-associated protein (isoform 1)
FLO9 166 1149 Lectin-like protein, flocculin (isoform 2)
LHS1 164 894 Chaperone
ZRT2 158 362 Zinc transport protein
SPF1 156 1217 P-type ATPase
ERG11 152 515 Lanosterol 14-alpha-demethylase
SSH1 147 479 Ssh1 translocon complex subunit
PMT1 144 789 Protein O-mannosyltransferase
ELO2 131 334 Fatty acid elongase
ERO1 126 527 Endoplasmic thiol oxidase
DCW1 123 448 Putative mannosidase
FRE2 117 716 Ferric reductase and cupric reductase (isoform 1)
SEC63 117 664 SEC63 complex (SEC63, SEC62, SEC66 and SEC72) subunit
PHM7 109 898 Uncharacterized phosphate metabolism protein
EMP70 108 620 Cellular adhesion and filamentous growth protein
MSB2 106 908 Mucin family member
Highest Expressed Posttranslationally Targeted Proteins
Gene cTPM Length Description
SCV12161.1 17336 55 Uncharacterized protein
SPI1 10900 135 GPI-anchored cell wall protein
CCA36283.1 10863 93 Uncharacterized protein
XP_002494332.1 10762 65 Uncharacterized protein
YNL190W 1856 132 Uncharacterized protein
AOA67261.1 568 233 Uncharacterized protein
AOA67185.1 211 221 Uncharacterized protein
AOA67185.1 209 201 Uncharacterized protein
AOA67185.1 209 197 Uncharacterized protein
AOA69306.1 205 229 Uncharacterized protein
LSM4 169 122 RNA processing Lsm protein
KRE1 127 95 Cell wall glycoprotein
FPR2 110 124 Peptidyl-prolyl cis-trans isomerase
FPR2 104 138 Peptidyl-prolyl cis-trans isomerase
MET16 98 287 3’-phosphoadenylsulfate reductase
SSP120 94 225 Uncharacterized protein
PRB1 84 559 Serine protease
AOA68412.1 66 375 Uncharacterized protein
LCL2 64 120 Uncharacterized long chronological lifespan protein
AOA67300.1 49 148 Uncharacterized protein
TIP1 17 194 Putative lipase
NPR3 9 789 SEA (SEH1-associated), NPR2/3, and IML1 complexes subunit
AOA68414.1 8 539 Putative arylsulfotransferase
NPR2 7 579 SEA (SEH1-associated), NPR2/3, and IML1 complexes subunit
YNL190W 5 193 Uncharacterized protein
AOA70062.1 4 523 Uncharacterized protein
FLO1 4 2435 Putative lectin-like protein
AOA70251.1 3 211 Uncharacterized protein
AOA68764.1 3 514 Uncharacterized protein
AOA66162.1 3 307 Putative Cu/Zn superoxide dismutase

Possible targets for S. cerevisiae

Highest Expressed Cotranslationally Targeted Proteins
Gene cTPM Length Description
PMA1 2369 919 Plasma membrane H+-ATPase
ECM33 1352 430 GPI-anchored cell wall organization protein Ecm33
SED1 1292 339 Major stress-induced structural GPI-cell wall glycoprotein
VMA3 1072 161 Vacuolar ATPase V0 domain V0 subunit c
PMP1 1006 41 ATPase proteolipid family
PMP2 999 44 ATPase proteolipid family
UTH1 944 366 Organelle organization
SCW4 876 387 Cell Wall
MRH1 857 321 Ion channel activity
SRL1 832 211 Mannoprotein that exhibits a tight association with the cell wall
GAS1 819 560 1,3-beta-glucanosyltransferase (isoform 2)
PHO84 782 588 Inorganic phosphate (Pi) transporter and low-affinity manganese transporter (isoform 1)
BGL2 769 314 Endo-beta-1,3-glucanase
PDI1 616 523 Protein disulfide isomerase
ERG25 594 310 C-4 methyl sterol oxidase
CCW14 589 239 Cell wall glycoprotein
KAR2 559 683 Protein import-associated ATPase
NCE102 555 174 Uncharacterized non-classical export protein
YNL190W 497 205 Uncharacterized protein
PHO12 469 468 Acid phosphatase
RTN1 437 296 Reticulon-like protein
TOS1 411 456 Uncharacterized protein
SIM1 399 477 Sun domain protein
CTS1 382 563 Endochitinase
OLE1 363 511 Delta(9) fatty acid desaturase (isoform 2)
ERG11 355 531 Lanosterol 14-alpha-demethylase
ERG2 342 223 C-8 sterol isomerase
ERG3 341 366 C-5 sterol desaturase
APE3 317 538 Aminopeptidase Y
PDR5 313 1512 Plasma membrane ATP-binding cassette (ABC) transporter
Highest Expressed Posttranslationally Targeted Proteins
Gene cTPM Length Description
CCW12 25439 134 Cell wall mannoprotein; plays a role in maintenance of newly synthesized areas of cell wall; localizes to periphery of small buds, septum region of larger buds, and shmoo tip; CCW12 has a paralog, YDR134C, that arose from the whole genome duplication
YDR524C-B 8301 67 Putative protein of unknown function; YDR524C-B has a paralog, YCL048W-A, that arose from the whole genome duplication
CWP2 3934 93 Covalently linked cell wall mannoprotein; major constituent of the cell wall; plays a role in stabilizing the cell wall; involved in low pH resistance; precursor is GPI-anchored
RPP2B 2437 111 60S ribosomal protein P2-alpha
HSP150 1581 414 Internal repeat-containing protein 1
CIS3 1016 228 Yeast PIR protein repeat
HOR7 698 60 Protein of unknown function; overexpression suppresses Ca2+ sensitivity of mutants lacking inositol phosphorylceramide mannosyltransferases Csg1p and Csh1p; transcription is induced under hyperosmotic stress and repressed by alpha factor; HOR7 has a paralog, DDR2, that arose from the whole genome duplication
PIR1 642 342 Internal repeat-containing protein 2
TOS6 243 103 Glycosylphosphatidylinositol-dependent cell wall protein; expression is periodic and decreases in respone to ergosterol perturbation or upon entry into stationary phase; depletion increases resistance to lactic acid
CWP1 220 240 Uncharacterized protein
TIP1 196 211 Putative lipase
PST2 191 199 Putative flavodoxin-like protein
KSH1 71 73 Transmembrane protein 167
AGA2 44 88 Adhesion subunit of a-agglutinin of a-cells; C-terminal sequence acts as a ligand for alpha-agglutinin (Sag1p) during agglutination, modified with O-linked oligomannosyl chains, linked to anchorage subunit Aga1p via two disulfide bonds
PIR3 25 326 Component of the outer cell wall layer. Required for stability of the cell wall and for optimal growth. Required for resistance against several antifungal and cell wall-perturbing agents
SUC2 21 533 Beta-fructofuranosidase
FIT3 19 205 Mannoprotein that is incorporated into the cell wall; incorporated via a glycosylphosphatidylinositol (GPI) anchor; involved in the retention of siderophore-iron in the cell wall
YEL068C 18 111 Protein of unknown function; expressed at both mRNA and protein levels
FIT2 17 154 Mannoprotein that is incorporated into the cell wall; incorporated via a glycosylphosphatidylinositol (GPI) anchor; involved in the retention of siderophore-iron in the cell wall
YHR138C 17 115 Protease B inhibitor
SWC7 10 133 Component of the SWR1 complex which mediates the ATP- dependent exchange of histone H2A for the H2A variant HZT1 leading to transcriptional regulation of selected genes by chromatin remodeling
DDR2 5 62 Multi-stress response protein; expression is activated by a variety of xenobiotic agents and environmental or physiological stresses; DDR2 has a paralog, HOR7, that arose from the whole genome duplication
KNH1 3 269 Cell wall synthesis protein
YCR101C 2 183 Functions as a sorting receptor in the Golgi compartment required for the intracellular sorting and delivery of soluble vacuolar proteins, like carboxypeptidase Y (CPY) and proteinase A. Executes multiple rounds of sorting by cycling between the late Golgi and a prevacuolar endosome-like compartment
SPI1 2 149 GPI-anchored cell wall protein
SPO1 1 632 Meiosis-specific prospore protein
PAU17 1 125 Member of the seripauperin multigene family encoded mainly in subtelomeric regions
GLG1 1 617 Glycogenin glucosyltransferase
YMR244W 1 356 Uncharacterized protein
PAU23 1 125 Member of the seripauperin multigene family encoded mainly in subtelomeric regions

S1: Nuclease footprinting and ribosome recovery

S2: Comparing demands from different species

References

“BRAR LAB — the Brar and Ünal Labs.” n.d. http://www.unal-and-brar-labs.org/brar-research/.

Dykstra, Karmen, Juho Rousu, and Mikko Arvas. 2017. “Predicting Protein Producibility in Filamentous Fungi.” bioRxiv.

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Huang, Mingtao, Guokun Wang, Jiufu Qin, Dina Petranovic, and Jens Nielsen. 2018. “Engineering the Protein Secretory Pathway of Saccharomyces Cerevisiae Enables Improved Protein Production.” Proc. Natl. Acad. Sci. U. S. A. 115 (47): E11025–E11032.

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